CD9+ Exosomes Open New Pathways for Extracellular Therapy in Salivary Gland Fibrosis

Chemical Reprogramming for Amplifying Salivary Gland Progenitor Cells

Researchers first isolated salivary gland basal progenitor cells (sgBPCs) from human parotid glands and developed a chemical reprogramming system using a combination of three small molecules (ROCK inhibitor, TGFβ inhibitor, and BMP inhibitor). This system not only preserves the epithelial characteristics of sgBPCs but also enables their expansion for over 50 passages in vitro while maintaining differentiation potential. Immunostaining revealed high expression of basal cell markers such as KRT5, KRT14, and SOX9, with minimal expression of terminal differentiation markers KRT7 and SOX2, confirming their undifferentiated state. In 3D matrices, these cells assembled into gland-like structures and differentiated into epithelial cell lineages, further validating their stemness. This system successfully established a stable and robust “factory” for EV production.

Fig.1 Schematic of sgBPCs isolation from human parotid glands

Microfluidic Chip-Based Isolation of High-Purity CD9⁺ EVs from sgBPCs

The next focus was on selecting the most effective products from this “factory.” Single-cell transcriptomic data revealed that the membrane protein CD9 is specifically highly expressed in basal cells, whereas CD63 and CD81 are predominantly found in non-epithelial cells. Inspired by this, researchers developed a CD9 antibody-based microfluidic sorting system, which, through affinity capture structures on the chip, isolates CD9-positive EVs (CD9⁺ EVs) with high purity in just 20 minutes.

Fig.2 Schematic of microfluidic device principle

Analysis showed that these EVs have a uniform size distribution, high purity, and 58.5% CD9 expression. They remained morphologically intact and maintained stable surface charge even after six months of cryopreservation, demonstrating the technical feasibility of precise EV isolation and laying the foundation for subsequent functional validation.

Fig.3 Expression and activity of CD9, CD81, and CD63 on CD9⁺ EVs after six months of storage

CD9⁺ EVs Exhibit Enhanced Uptake and Enrichment of Regeneration-Related Proteins

With a stable source and reliable sorting method, researchers investigated whether CD9⁺ EVs met therapeutic expectations. Uptake experiments showed that CD9⁺ EVs were more readily internalized by salivary gland epithelial cells, with significantly higher uptake compared to CD9⁻ EVs and mixed EV groups. Mass spectrometry revealed that CD9⁺ EVs are enriched with proteins related to membrane fusion, cell adhesion, and tissue repair, such as TSPAN4, CD81, CD82, and integrins ITGA3/ITGB1—key signaling components in extracellular communication and repair processes.

Fig.4 Uptake of CD9⁺ exosomes by salivary epithelial cells and CD9 protein interaction network

Notably, CD9 directly interacts with CD44, a common epithelial adhesion molecule, which may explain its efficient uptake by target cells. In contrast, CD9⁻ EVs primarily carry immune chemotaxis-related contents, making them less suitable for tissue regeneration.

CD9⁺ EVs Reverse Tissue Damage in a Mouse Fibrosis Model

To evaluate the in vivo efficacy of CD9⁺ EVs, researchers established a mouse salivary gland duct ligation model, inducing severe fibrosis through 14 days of ligation. EVs were then locally injected into the gland via the duct.

Fig.5 Mouse salivary gland duct ligation model

The results were promising: compared to PBS and CD9⁻ EV groups, the CD9⁺ EV-treated group showed significant improvements in salivary secretion rate, onset time, and gland weight recovery. Histological staining (H&E, PAS) revealed better restoration of acinar and ductal structures, while MTC and Sirius Red staining indicated significantly reduced collagen deposition and alleviated fibrosis. Remarkably, CD9⁺ EVs retained significant therapeutic efficacy even after six months of cryopreservation, highlighting their stability and clinical potential.

Fig.6 Representative images of salivary glands one week after ligation removal and EV treatment

Fig.7 Comparison of parameters after ligation removal

Mechanistic Insights: CD9⁺ EVs Suppress EMT and Promote Progenitor Cell Proliferation

To elucidate the mechanism of action, researchers observed changes in epithelial cells. In the CD9⁺ EV-treated group, progenitor and mature acinar cell marker expression was restored, cell proliferation was significantly enhanced, and epithelial-mesenchymal transition (EMT) markers such as CDH2, VIM, SLUG, and TWIST1 were suppressed. In other words, CD9⁺ EVs not only promoted functional epithelial cell proliferation and differentiation but also effectively inhibited fibrosis progression.

Further validation was conducted using an in vitro organoid model. Salivary gland organoids were induced to undergo fibrotic changes with Activin A, then treated with CD9⁻ EVs, CD9⁺ EVs, or dexamethasone as a positive control. Results showed that CD9⁺ EVs significantly outperformed other groups in restoring organoid structure and function. qPCR analysis revealed downregulation of fibrosis-related genes such as COL1A1 and SNAI1, alongside restoration of secretory markers. A critical breakthrough came from miRNA sequencing, which showed that CD9⁺ EVs are specifically enriched with miR-3162 and miR-1290, both of which directly target ACVR1 (an Activin receptor), thereby blocking SMAD2/3 phosphorylation—a key fibrosis pathway. Reporter gene assays confirmed the existence of this mechanistic axis, completing the mechanistic loop.

Fig.8 Organoid model experimental results

This study systematically explores the therapeutic potential of extracellular vesicles, establishing a highly logical and practical research pathway. From foundational cell establishment to precise purification technology development, in vivo and in vitro efficacy validation, and mechanistic elucidation, it comprehensively demonstrates the role of extracellular vesicles in anti-fibrotic therapy. The findings highlight that CD9⁺ EVs offer technical advantages for scalable production and cryopreservation, alongside robust bioactivity and clinical translation potential, underscoring the importance of “selecting the right signaling carrier to deliver effective biological information” in regenerative medicine.

CD9 is a widely expressed tetraspanin family protein found on the surface of various cell types. Comprising approximately 228 amino acids, it features four transmembrane domains, two extracellular loops (large and small), and an intracellular tail. Highly conserved, CD9 is expressed in immune cells, epithelial cells, platelets, and various tumor cells, with key functions including:

• Facilitating cell-cell and cell-matrix adhesion and interactions
• Regulating cell migration and tissue remodeling
• Participating in cell fusion processes
• Serving as a marker protein for extracellular vesicles, mediating EV formation and information transfer

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